Curcumin-rich plant material production through a robust and reproducible micropropagation protocol.

Curcuma longa Linné is a widely valued spice due to its nutritional and therapeutic properties. Its production methods, vulnerability to diseases, and increasing anthropogenic pressure contribute to the limited availability of planting materials. This study aims to develop a micropropagation protocol that ensures the production of curcumin-rich planting stocks. Rhizomes were either pre-treated or not with Copper hydroxide (8%), disinfected with Mercuric chloride (HgCl₂ at 0.5%, 1%, or 2%), then treated with Ethanol (70%), followed by disinfection with Sodium hypochlorite (NaClO at 30%, 40%, or 50%). Buds from primary and secondary rhizomes were cultured on Murashige and Skoog medium supplemented with 6-benzylaminopurine (2 mg/L or 4 mg/L), Kinetin (4 mg/L), and Magnesium chloride (0.75 g/L). Data were analyzed using ANOVA, and a linear model was applied to determine the relationships between parameters and treatments. The optimal disinfection protocol that minimized contamination and necrosis while promoting successful in vitro establishment consisted of HgCl₂ (0.1%), NaClO (30%), and Ampicillin (0.1%). The addition of Magnesium chloride (MgCl₂) to the culture medium enhanced organogenesis and proliferation, with significant improvement when combined with BAP (4 mg/L), resulting in increased shoot proliferation (15.4 shoots per plant), root number (12.1 ± 0.15), leaf development (11.3 ± 0.08), as well as improved shoot elongation (12.38 ± 1.4 cm) and root elongation (5.18 ± 0.5 cm). During acclimatization, the "Terreau Semis Pouss'activR U" (TSP) substrate ensured optimal survival (100%) and better shoot and leaf growth compared to the local substrate composed of sand and compost (1:1) and the SAH substrate based on moderately decomposed sphagnum peat and black peat. This optimized protocol supports large-scale of curcumin-rich plant material production and enhanced valorization of curcuma.